Congenital heart defects (CHDs) affect almost 1% of all live human births and frequently require surgical intervention in order to prevent death. The causes of these CHDs frequently involve the disregulation of events within the transcriptional programs that control cardiac specification, patterning, differentiation and morphogenesis. Hand1 and Hand2 are evolutionary conserved basic Helix-Loop-Helix (bHLH) transcription factors that exhibit partially overlapping spatiotemporal expression patterns during cardiac development. Hand1 and Hand2 are initially co-expressed, but following looping;Hand1 is predominantly restricted to the left ventricle, whereas Hand2 is predominantly restricted to the right ventricle. However, both genes remain co-expressed in the aortic sac, outflow tract (OFT) and ventricular expression borders of the interventricular septum. Our data show that unlike most Class B bHLH proteins, Hand factors can utilize a wide range of bHLH partners (E-proteins, Twist-family, and Hrts). We furher show that a highly conserved phosphoregularty circuit controls dimer-choice and that the disregulation of phosphorylation can result in the human disease Saethre-Chotzen Syndrome. From these data, we hypothesize a mechanism whereby three parameters will dictate Hand factor function in the heart. First is partner choice/availability, second is the phosphorylation state of Helix I residues, and third is the overall transcriptional regulation of Hand factors within a given cardiomyocyte. To test the roles of these parameters, we have formulated three-Specific Aims. Specific Aim 1 will test partner choice/availability by altering the gene dosage of Hand2 in the cells that express Hand1. Specific Aim 2 will test the role of Hand1 Helix 1 phosphorylation by conditional knock-in of hypophosphorylation and phosphorylation mimic proteins. Specific Aim 3 will address the transcriptional regulation of Hand1 via Nkx2.5 and Mef2c, which we show can genetically interact during cardiogenesis. Taken together, these aims will extend and refine our current understanding of Hand factor function during cardiogenesis and directly address the questions of functional redundancy (a function of partner choice/availability), the role of Handl phosphorylation in heart development, and the role of transcription factors upstream of Handl in defining the overall level of Handl protein in the cardiomyocyte. The understanding gained from this proposal will add significant insight into the molecular mechanisms that drive heart formation and go awry in human disease.